other choice of TSK5000PWxl by TosoHaas

[kurian]

I would like to change my column for an other because my peaks are retained in Vo.
I have use Ultrahydrogel Linear 250 from Waters and Aquagel-OH 50 from Polymer lab.

Someone know a good Column for conjugates Polysaccharide-Carrier in Molecular Size Distribution ?

[Uwe Neue]

Many questions:
What do you mean by the statement that your peak are retained in Vo? What is the elution time at what flow rate for which column (with column dimensions)?
What are the mobile phases that you have worked with?
What is a "conjugates poylsaccharide carrier", and what is the molecular weight that you are expecting to see?

[kurian]

Mobile phase : PO4 50 mM, NaCl 200 mM at pH 7.00
I can't change the phase, NaCl 0.2M certainly not and that's why my recovery on column is very low

I work in isocratic with a flow 0.75 ml/min

The elution time for the first peak is 6 min, second 8.5 min and thirth 12.5 min

The samples are "conjugated" in Polysaccharide form with protein content.
I wait for reply to have structure of samples and molecular weight.

[Uwe Neue]

The separation space for the Ultrahydrogel column under your conditions is between about 6 minutes to about 14.5 minutes. Your first peak is excluded. If you want to get a molecular weigt distribution for the first peak, you need to select a larger pore size. In order to select the right pore size, you need to know at least something about the molecular weight of this compound.

How do you know that your recovery is low?

[kurian]

Just calculate the total area of peaks on column and make the division with total area without column on refractometre.
The calcul give 22 % of recovery on column Aquagel

For the instant, Ultrahydrogel gives the bests results( 40%), but we want more.
Same results for TSK5000PWxl but problem of reproducibility with other batch columns (evolution of Dextran 3 800 000 Da in Vo or not).

The molecular size of sample is about 350 000 Da. His structure is linear, like a spaghetti

Here an image of the structure :

[Uwe Neue]

For multiple reasons, I am not convinced that the peak area that you get from the refractometer is a good reference point. What was the flow rate that you used for this experiment?

The Ultrahydrogel 250 is too small for such an analysis: your peak is excluded from the pores. My suggestion: buy an Ultrahydrogel Linear column, and at least you get some idea of the MW of your analytes. This may actually be sufficient for the analysis that you need to do. But just an excluded peak does not give you any information, except that there is a peak.

[Rande]

I have worked with HMW conjugate polysaccharides in the past. I think we used Sephacryl S 400 or S500. This might only be available for Prep scale, I am not sure what GE has available for analytical.

I would suggest the TSK5000PWxl , you said you were having problems with that column. How are your system suitability tests for your column? Especially with SEC - you MUST run system suitability tests with every assay run (I do - before and after samples).

A few random thoughts:

- are you sure about the conjugate being linear? the polysaccharide subunit might be, but due to cross linking with the protein conjugation -it can have multiple branching. This is dependent on the conjugation chemistry used and the ratio of protein to polysaccharide.

- Are you only using RI detector? you might be able to see the protein - conjugate with UV. Of course the unconjugated polysaccaride would probably not be detected by UV.

[kurian]

of course the conjugate is not very linear cause the structure with proteins, but it's not a globular molecule.

My flow rate is 0.75 ml/min
the real problem with TSK5000P is the Dextran 3 800 000 Da. Sometimes a part of particle is eluated in Vo , sometimes all of Dextran is in elution volume.
This Dextran is MY REFERENCE FOR ALL TESTING, representing the reproducibility column by column.
I use for all my RUN (or Sequence) a suitability test, Azide 0.025% at 100 µl injected.

We search an alternative, such as Ultrahydrogel 250

Of course i use a detector DAD in line with RI.

Rande have used in the past columns Sephacryl S400 or S500 --> Column with Silicium structure is best for Molecular Size Distribution ?

[Uwe Neue]

Very large random molecules can be subject to degradation by shear. By that I mean that you can get different results depending on how quickly you (or your injector) suck(s) the sample into the loop and related things. Also, the age of the sample may play a role.

I do not understand, why you think that the UltraHydrogel 250 and the TSK 5000P have the same pore size, and would give you equivalent results.

I still think that a Ultrahydrogel Linear column will serve you best. It has a very wide pore size distribution, and no matter what your size is, you will get some retention.

[kurian]

ok,

thanks